1. Field
The invention is concerned with novel water soluble N-(4-carbamimidoyl-phenyl)-glycine derivatives.
2. Description
Inhibitors of factor VIIa have been suggested for the inhibition of the formation of thrombi and for the treatment of related diseases (WO 00/35858). Unfortunately, the compounds disclosed in WO 00/35858 have lower than desirable solubility in water and are not particularly suitable for subcutaneous applications.
Accordingly, there is a long felt need in the art for factor VIIa inhibitors that have increased water solubility and enhanced inhibitory activity.
The subject invention provides compounds of the formula: 
wherein
R1 is lower-alkoxy;
R2 is halogen;
R3 is piperidinyl or piperidinyl substituted with lower-alkyl, hydroxy-lower-alkyl, carboxy-lower-alkyl or lower-alkoxy-carbonyl-lower-alkyl;
R4 is hydrogen or lower-alkyl;
A is xe2x80x94(CH2)nxe2x80x94 or xe2x80x94(CH2)nxe2x80x94 which is substituted with lower-alkyl; and
n is 0 to 3. Pharmaceutically acceptable salts of these compounds are also an aspect of this invention.
Certain preferred compounds are where R1 is ethoxy, R2 is fluorine, and R3 is piperidinyl or piperidinyl substituted with methyl or hydroxy-ethyl, such as 1-methyl-piperidin-3-yl; 1-(2-hydroxy-ethyl)-piperidin-4-yl; 1-(2-hydroxy-ethyl)-piperidin-3-yl; piperidin-4-yl; or 1-methyl-piperidin-4-yl. R4 is favorably hydrogen or ethyl, and especially hydrogen. It is preferred where n is 0 to 2, and favorably n is 0.
A preferred group of compounds are of the formula 
wherein R1, R2, R3, R4, A, and n have the significances given above. As above, pharmaceutically acceptable salts of these compounds are also an aspect of this invention.
Other useful compounds are of the formula: 
wherein R1, R2, R3, R4, A, and n have the significances given above. Pharmaceutically acceptable salt of these compounds are also an aspect of the invention.
The subject invention will now be described in terms of its preferred embodiments. These embodiments are set forth to aid in understanding the invention but are not to be construed as limiting the invention in any way.
The invention is concerned with water soluble N-(4-carbamimidoyl-phenyl)-glycine derivatives of the formula (I) 
wherein
R1 is lower-alkoxy,
R2 is halogen,
R3 is piperidinyl optionally substituted with lower-alkyl, hydroxy-lower-alkyl, carboxy-lower-alkyl or lower-alkoxy-carbonyl-lower-alkyl,
R4 is hydrogen or lower-alkyl,
n is 0 to 3, and the xe2x80x94(CH2)nxe2x80x94 group can optionally be substituted with lower-alkyl, and pharmaceutically acceptable salts thereof.
Further, the invention is concerned with a process for the manufacture of the above compounds, pharmaceutical preparations which contain such compounds as well as the use of these compounds for the production of pharmaceutical preparations.
The compounds of formula (I) are active compounds and inhibit the formation of coagulation factors Xa, Ixa and thrombin induced by factor VIIa and tissue factor or are derivatives which are converted under physiological conditions to such active compounds. These compounds consequently influence both platelet aggregation which is induced by these factors and plasmatic blood coagulation. They therefore inhibit the formation of thrombi and can be used for the treatment and/or prevention of diseases, such as arterial and venous thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina pectoris, cardiac infarction, stroke due to atrial fibrillation, inflammation and arteriosclerosis. Furthermore, these compounds have an effect on tumor cells and prevent metastases. They can therefore also be used as antitumor agents.
Inhibitors of factor VIIa had previously been suggested for the inhibition of the formation of thrombi and for the treatment of related diseases (WO 00/35858). However, there is still a need for factor VIIa inhibitors which exhibit a much higher solubility in water in order to be suitable for subcutaneous applications and which at the same time exhibit a higher inhibitory activity.
The present invention provides compounds of formula (I) which are factor VIIa inhibitors and unexpectedly exhibit the desired increased water solubility and increased inhibitory activity compared to the compounds known from WO 00/35858. These inventive compounds further exhibit improved pharmacological properties compared to the known compounds.
Unless otherwise indicated, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention herein.
In this specification the term xe2x80x9clowerxe2x80x9d is used to mean a group consisting of one to seven, preferably of one to four carbon atom(s).
The term xe2x80x9chalogenxe2x80x9d refers to fluorine, chlorine, bromine and iodine, with fluorine, chlorine and bromine being preferred.
The term xe2x80x9calkylxe2x80x9d, alone or in combination with other groups, refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of one to twenty carbon atoms, preferably one to sixteen carbon atoms, more preferably one to ten carbon atoms. Lower-alkyl groups as described below also are preferred alkyl groups.
The term xe2x80x9clower-alkylxe2x80x9d, alone or in combination with other groups, refers to a branched or straight-chain monovalent alkyl radical of one to seven carbon atoms, preferably one to four carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.
The term xe2x80x9calkoxyxe2x80x9d refers to the group Rxe2x80x2xe2x80x94Oxe2x80x94, wherein Rxe2x80x2 is an alkyl. The term xe2x80x9clower-alkoxyxe2x80x9d refers to the group Rxe2x80x2xe2x80x94Oxe2x80x94, wherein Rxe2x80x2 is a lower-alkyl.
Compounds of formula (I) can form pharmaceutically acceptable acid addition salts. Examples of such pharmaceutically acceptable salts are salts of compounds of formula (I) with physiologically compatible mineral acids, such as hydrochloric acid, sulfuric acid, sulphurous acid or phosphoric acid; or with organic acids, such as methanesulphonic acid, p-toluenesulphonic acid, acetic acid, lactic acid, trifluoroacetic acid, citric acid, fumaric acid, maleic acid, tartaric acid, succinic acid or salicylic acid. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to such salts. Compounds of formula (I) can further form salts with bases. Examples of such salts are alkaline, earth-alkaline and ammonium salts such as e.g. Na-, K-, Ca- and Trimethylammonium salt. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d also refers to such salts.
In detail, the present invention relates to compounds of formula (I) 
wherein
R1 is lower-alkoxy,
R2 is halogen,
R3 is piperidinyl optionally substituted with lower-alkyl, hydroxy-lower-alkyl, carboxy-lower-alkyl or lower-alkoxy-carbonyl-lower-alkyl,
R4 is hydrogen or lower-alkyl,
n is 0 to 3, and the xe2x80x94(CH2)nxe2x80x94 group can optionally be substituted with lower-alkyl, and pharmaceutically acceptable salts thereof.
The compounds of formula (I) have at least,one asymmetric C atom and can therefore exist as an enantiomeric mixture, diastereomeric mixture or as optically pure compounds. Compounds of formula (I) can exist in tautomeric forms and the invention encompasses all such tautomeric forms.
Compounds of formula (I) are individually preferred and physiologically acceptable salts thereof are individually preferred, with the compounds of formula (I) being particularly preferred.
Preferred compounds of formula (I) are those, wherein R1 is ethoxy. Another preferred embodiment of the present invention relates to compounds as described above, wherein R2 is fluorine.
In a further preferred embodiment the invention relates to compounds as described above in which R3 is piperidinyl optionally substituted with methyl or hydroxy-ethyl. Compounds in which R3 is 1-methyl-piperidin-3-yl, 1-(2-hydroxy-ethyl)-piperidin-4-yl, 1-(2-hydroxy-ethyl)-piperidin-3-yl, piperidin-4-yl or 1-methyl-piperidin-4-yl are more preferred.
The invention embraces especially compounds in accordance with the above definitions in which R4 is hydrogen or ethyl, with those wherein R4 is hydrogen being more preferred.
Moreover, the invention relates especially to compounds as defined above in which n is 0-2, with those wherein n is 0 being particularly preferred.
Of the compounds of formula (I) as described above, the R-enantiomers are preferred. Such R-enantiomers are characterised by formula (Ia). Therefore compounds according to the definitions given above characterised by formula (Ia) 
wherein R1, R2, R3, R4 and n have the significances given above, are also preferred.
In particular, preferred compounds are the compounds of formula (I) described in the examples as individual compounds in the form of the free acids, their esters as well as hydrates or solvates and pharmaceutically acceptable salts thereof.
Preferred compounds of formula (I) are those selected from the group consisting of
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-[(RS)-1-methyl-piperidin-3-ylmethoxy]-phenyl]-acetic acid ethyl ester hydrochloride,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-[(RS)-1-methyl-piperidin-3-ylmethoxy]-phenyl]-acetic acid,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-[(RS)-1-methyl-piperidin-3-yloxy]-phenyl]-acetic acid ethyl ester hydrochloride,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-[(RS)-1-methyl-piperidin-3-yloxy]-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(2-piperidin-1-yl-ethoxy)-phenyl]-acetic acid ethyl ester hydrochloride,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(2-piperidin-1-yl-ethoxy)-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(2-piperidin-4-yl-ethoxy)-phenyl]-acetic acid ethyl ester hydrochloride,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(2-piperidin-4-yl-ethoxy)-phenyl]-acetic acid,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-[(RS)-1-methyl-2-piperidin-1-yl-ethoxy]-phenyl]-acetic acid ethyl ester hydrochloride,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-[(RS)-1-methyl-2-piperidin-1-yl-ethoxy]-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(1-methyl-piperidin-4-ylmethoxy)-phenyl]-acetic acid ethyl ester acetate,
(RS)-(4-Carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(1-methyl-piperidin-4-ylmethoxy)-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(piperidin-4-ylmethoxy)-phenyl]-acetic acid ethyl ester acetate,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(piperidin-4-ylmethoxy)-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-3-(1-ethoxycarbonylmethyl-piperidin-4-yloxy)-2-fluoro-phenyl]-acetic acid ethyl ester hydrochloride,
(RS)-(4-carbamimidoyl-phenylamino)-[3-(1-carboxymethyl-piperidin-4-yloxy)-5-ethoxy-2-fluoro-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-{5-ethoxy-2-fluoro-3-[1-(2-hydroxy-ethyl)-piperidin-4-yloxy]-phenyl}-acetic acid ethyl ester hydrochloride,
(RS)-(4-carbamimidoyl-phenylamino)-{5-ethoxy-2-fluoro-3-[1-(2-hydroxy-ethyl)-piperidin-4-yloxy]-phenyl}-acetic acid,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-3-[(RS)-1-ethoxycarbonylmethyl-piperidin-3-yloxy]-2-fluoro-phenyl]-acetic acid ethyl ester hydrochloride,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[3-[(RS)-1-carboxymethyl-piperidin-3-yloxy]-5-ethoxy-2-fluoro-phenyl]-acetic acid,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-{5-ethoxy-2-fluoro-3-[(RS)-1-(2-hydroxy-ethyl)-piperidin-3-yloxy]-phenyl}-acetic acid ethyl ester hydrochloride,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-{5-ethoxy-2-fluoro-3-[(RS)-1-(2-hydroxy-ethyl)-piperidin-3-yloxy]-phenyl}-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(piperidin-4-yloxy)-phenyl]-acetic acid ethyl ester hydrochloride,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(piperidin-4-yloxy)-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(1-methyl-piperidin-4-yloxy)-phenyl]-acetic acid ethyl ester hydrochloride(1:2),
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(1-methyl-piperidin-4-yloxy)-phenyl]-acetic acid,
(S)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(1-methyl-piperidin-4-yloxy)-phenyl]-acetic acid, and
(R)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(1-methyl-piperidin-4-yloxy)-phenyl]-acetic acid, and pharmaceutically acceptable salts thereof.
Particularly preferred compounds of formula (I) are those selected from the group consisting of
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-[(RS)-1-methyl-piperidin-3-yloxy]-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-{5-ethoxy-2-fluoro-3-[1-(2-hydroxy-ethyl)-piperidin-4-yloxy]-phenyl}-acetic acid,
(RS)- and (SR)-(4-carbamimidoyl-phenylamino)-{5-ethoxy-2-fluoro-3-[(RS)-1-(2-hydroxy-ethyl)-piperidin-3-yloxy]-phenyl}-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(piperidin-4-yloxy)-phenyl]-acetic acid,
(RS)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(1-methyl-piperidin-4-yloxy)-phenyl]-acetic acid, a (R)-(4-carbamimidoyl-phenylamino)-[5-ethoxy-2-fluoro-3-(1-methyl-piperidin-4-yloxy)-phenyl]-acetic acid, and pharmaceutically acceptable salts thereof
It will be appreciated that the compounds of general formula (I) in this invention may be derivatised at functional groups to provide derivatives which are capable of conversion back to the parent compound in vivo.
The invention further relates to a process for the manufacture of compounds of formula (I) as defined above, which process comprises converting the nitrile group in a compound of formula (II) 
wherein R1, R2, R3, R4 and n have the significances given above, into a carbamimidoyl group and, if desired, converting an obtained compound of formula (I) into a pharmaceutically acceptable salt or converting a salt of a compound of formula (I) into the corresponding compound of formula (I). A preferred process as described above comprises the conversion of the nitrile group into a carbamamidoyl group and, if desired, converting an obtained compound of formula (I) into a pharmaceutically acceptable salt, more preferably the conversion of the nitrile group into a carbamamidoyl group.
The conversion of the nitrile group in a compound of formula (II) into a carbamimidoyl-group xe2x80x94C(NH)NH2 can be carried out according to methods known per se.
The conversion of the nitrile group into a carbamimidoyl group can be carried out e.g. by treating a compound of formula (II) in a solvent, such as ethanol or methanol, or a solvent mixture, such as chloroform and methanol or chloroform and ethanol, with a dry stream of hydrogen chloride, conveniently at a temperature below 10xc2x0 C., thereafter treating the reaction solution with a solvent, such as diethyl ether, and filtering off the precipitated iminoether. The thus-obtained material is treated in a solvent, such as methanol or ethanol, either with gaseous ammonia or an ammonium salt, such as ammonium acetate, conveniently at a temperature up to 80xc2x0 C. Alternatively, the solution containing the iminoether can be evaporated and the residue can be treated with gaseous ammonia or an ammonium salt in methanol or ethanol.
The conversion of the nitrile group in a compound of formula (II) into a carbam-imidoyl group can also be achieved by conversion of the nitrile group into a N-hydroxy-carbamimidoyl group and subsequent reduction. For example, the conversion into a N-hydroxy-carbamimidoyl group can be performed by dissolving a compound of formula (II) in a solvent, such as DMF, ethanol or methanol, treating the solution with hydroxyl-amine or a salt of hydroxylamine with an inorganic acid, such as hydroxylamine hydrochloride, and thereafter with a base, such as diisopropylethylamine or triethylamine, sodium hydride or sodium methanolate, conveniently at a temperature up to 80xc2x0 C. For the conversion of a N-hydroxy-carbamimidoyl group into a carbamimidoyl group, the compound can be hydrogenated in a solvent, such as ethanol, methanol, dioxan, THF or glacial acetic acid, or a solvent mixture, such as ethanol and glacial acetic acid, with hydrogen and a catalyst, such as palladium, platinum or nickel. In so doing, other reactive groups present in the compound of formula I and reactive towards the reducing agent can be modified.
An ester group (R4=lower-alkyl) can be converted to the carboxy group by methods known in the art, e.g. by hydrolysis with sodium hydroxide in water or water/THF at a temperature in the range of 0 to 30xc2x0 C. during 1 to 5 hours.
The compounds of formula (II) can be prepared according to general methods known per se, e.g. as described hereinafter and/or as described in the Examples or in analogy to these methods. For example, a compound of formula (III) 
in which R1, R2 and R4 have the significances given above and R5=H can be reacted: with an alkylating agent such as an appropriately substituted alkyl bromide, alkyl iodide or alkyl mesylate in the presence of a base such as potassium carbonate or caesium carbonate in a solvent such as DMF or acetone, or by a Mitsunobu reaction with an appropriately substituted alkohol in the presence of DEAD, DIAD, or di-tert.-butyl-azodicarboxylate, and triphenylphosphine in a solvent such as THF or dioxane, there are obtained compounds of formula (II)
Suitable piperidinyl-substituted alkyl halogenides or alcohols, which are starting materials for such a reaction, are either commercially available or can be prepared by methods known in the art or in analogy to the examples described.
A compound of formula (III) in which R5 represents hydrogen can be obtained from a compound of formula (III) in which R5 represents a protecting group (e.g. a benzyl group, an isopropyl group or a tert-butyl dimethylsilyl group) by methods known per se, see T. W. Greene, P.G.M Wuts xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d, 2nd ed., John Wiley and Sons, Inc., New York/Chichester/Brisbane/Toronto/Singapore 1991.
The compounds of formula (III) can be prepared according to general methods known per se, e.g. as described hereinafter and/or as described in the Examples or in analogy to these methods. For example, an aldehyde of formula (IV) 
in which R1 and R2 have the significances given above and R5 represents hydrogen or a protecting group (e.g. a benzyl group, an isopropyl group or a tert-butyl dimethylsilyl group) can be reacted with a p-aminobenzonitrile of formula (V) 
and benzylisonitrile, toluenesulfonylmethylisonitrile, or morpholinoethylisocyanide, and a primary alkanol such as methanol or ethanol, in the presence of boron trifluoride etherate. Hydrolysis of the resulting iminoether with water yields a compound of formula (III) in which R4 represents methyl or ethyl and R5 represents hydrogen or a protecting group (e.g. a benzyl group, an isopropyl group or a tert-butyl dimethylsilyl group). By hydrolysis of the ester group R4, e.g. by treatment with LiOH in tetrahydrofuran, there is obtained a compound of formula (III) in which R4 represents hydrogen.
Compounds of formula (IV) are known per se or can be prepared according to methods known per se, e.g. as described hereinafter and/or as described in the Examples or in analogy to these methods.
Starting materials for the preparation of compounds of formula (IV) are either commercially available or can be prepared by methods known in the art.
Insofar as their preparation is not described in the examples, the compounds of formulae (I), (II), (III) and (IV) can be prepared according to analogous methods or according to the methods set forth above.
Furthermore, the invention relates to compounds of formula (I) as defined above, when manufactured by a process as described above. In another embodiment, the invention relates to the intermediates, the compounds of formula (II) 
wherein R1, R2, R3, R4 and n have the significances given above.
As described above, the compounds of formula (I) are active compounds and inhibit the formation of coagulation factors Xa, IXa and thrombin induced by factor VIIa and tissue factor or are derivatives which are converted under physiological conditions to such active compounds. These compounds consequently influence both platelet aggregation which is induced by these factors and plasmatic blood coagulation. They therefore inhibit the formation of thrombi and can be used for the treatment and/or prevention of diseases, such as arterial and venous thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina pectoris, cardiac infarction, stroke due to atrial fibrillation, inflammation and arteriosclerosis. Furthermore, these compounds have an effect on tumor cells and prevent metastases. They can therefore also be used as antitumor agents. Prevention and/or treatment of thrombosis is the preferred indication.
The invention therefore also relates to pharmaceutical compositions comprising a compound as defined above and a pharmaceutically acceptable carrier and/or adjuvant.
The invention likewise embraces compounds as described above for use as therapeutically active substances, especially as therapeutically active substances for the treatment and/or prophylaxis of diseases which are associated with the formation of clotting factors Xa, IXa and thrombin induced by factor VIIa and tissue factor, particularly as therapeutically active substances for the treatment and/or prophylaxis of arterial and venous thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina pectoris, cardiac infarction, stroke due to atrial fibrillation, inflammation, arteriosclerosis and/or tumor.
In another preferred embodiment, the invention relates to a method for the therapeutic and/or prophylactic treatment of diseases which are asscociated with the formation of clotting factors Xa, IXa and thrombin induced by factor VIIa and tissue factor, particularly for the therapeutic and/or prophylactic treatment of arterial and venous thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina pectoris, cardiac infarction, stroke due to atrial fibrillation, inflammation, arteriosclerosis and/or tumor, which method comprises administering a compound as defined above to a human being or animal.
The invention also embraces the use of compounds as defined above for the therapeutic and/or prophylactic treatment of diseases which are asscociated with the formation of clotting factors Xa, IXa and thrombin induced by factor VIIa and tissue factor, particularly for the therapeutic and/or prophylactic treatment of arterial and venous thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina pectoris, cardiac infarction, stroke due to atrial fibrillation, inflammation, arteriosclerosis and/or tumor.
The invention also relates to the use of compounds as described above for the preparation of medicaments for the therapeutic and/or prophylactic treatment of diseases which are asscociated with the formation of clotting factors Xa, IXa and thrombin induced by factor VIIa and tissue factor, particularly for the therapeutic and/or prophylactic treatment of arterial and venous thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina pectoris, cardiac infarction, stroke due to atrial fibrillation, inflammation, arteriosclerosis and/or tumor. Such medicaments comprise a compound as described above.
The inhibition of the amidolytic activity of factor VIIa/tissue factor complex by the compounds in accordance with the invention can be demonstrated with the aid of a chromogenic peptide substrate as described hereinafter.
The measurements were carried out on microtiter plates at room temperature. To this end, 100 xcexcl of a solution of 26 nM of tissue factor, 9 nM of soluble factor VIIa and 8 mM of calcium chloride were added to 25 xcexcl of a solution of the inhibitor in a buffer [pH 7.5, 100 mM, comprising 0.14M NaCl, 0.1M N-(2-hydroxyethyl)piperadine-Nxe2x80x2-(2-ethanesulphonic acid) (HEPES), 0.5 mg/l of fatty-acid-free BSA (bovine serum albumin) and 0.05% NaN3] in each well of the plate. After an incubation time of 15 minutes the reaction was started by the addition of 50 xcexcl of chromogenic substrate Chromozym-tPA (3.5 mM, MeSO2-D-Phe-Gly-Arg-paranitroanilide) and the hydrolysis of the substrate was followed spectrophotometrically on a kinetic microtiter plate reader over 10 minutes. Using the plot of the inhibition curves, the Ki values were determined according to the method described in Biochem. J. 55, 1953, 170-171.
The activity of the low molecular weight substances can, moreover, be characterized in the xe2x80x9cprothrombixe2x80x9d timexe2x80x9d (PT) clotting test. The substances are prepared as a 10 mM solution in DMSO or DMSO/0.1M HCl (DHCl) and thereafter made up to the desired dilution in the same solvent. Thereafter, 0.25 ml of human plasma (obtained from whole blood anticoagulated with 1/10 volume of 108 mM Na citrate) was placed in the instrument-specific sample container. In each case 5 xcexcl of each dilution of the substance-dilution series was then mixed with the plasma provided. This plasma/inhibitor mixture was incubated at 37xc2x0 C. for 2 minutes. Thereafter, there were pipetted to the semi-automatic device (ACL, Automated Coagulation Laboratory (Instrument Laboratory)) 50 xcexcl of plasma/inhibitor mixture in the measurement container. The clotting reaction was initiated by the addition of 0.1 ml of Innovin(copyright) (recombinant human tissue factor combined with calcium buffer and synthetic phospholipids(Dade Behring(copyright), Inc.). The time up to the fibrin cross-linking was determined photooptically from the ACL. The inhibitor concentration, which brought about a doubling of the PT clotting time, was determined by means of a graph.
The Ki value of the active compounds of the present invention preferably amounts to about 0.1 to 500 nM, especially about 0.1 to 150 nM. The PT values preferably amount to about 0.1 to 10 xcexcM, especially to about 0.1 to 5 xcexcM.
The compounds of formula I and/or their pharmaceutically acceptable salts can be used as medicaments, e.g. in the form of pharmaceutical preparations for enteral, parenteral or topical administration. They can be administered, for example, perorally, e.g. in the form of tablets, coated tablets, dragxc3xa9es, hard and soft gelatine capsules, solutions, emulsions or suspensions, rectally, e.g. in the form of suppositories, parenterally, e.g. in the form of injection solutions or suspensions or infusion solutions, or topically, e.g. in the form of ointments, creams or oils. Parenteral administration is preferred.
The production of the pharmaceutical preparations can be effected in a manner which will be familiar to any person skilled in the art by bringing the described compounds of formula I and/or their pharmaceutically acceptable salts, optionally in combination with other therapeutically valuable substances, into a galenical administration form together with suitable, non-toxic, inert, therapeutically compatible solid or liquid carrier materials and, if desired, usual pharmaceutical adjuvants.
Suitable carrier materials are not only inorganic carrier materials, but also organic carrier materials. Thus, for example, lactose, corn starch or derivatives thereof, talc, stearic acid or its salts can be used as carrier materials for tablets, coated tablets, dragxc3xa9es and hard gelatine capsules. Suitable carrier materials for soft gelatine capsules are, for example, vegetable oils, waxes, fats and semi-solid and liquid polyols (depending on the nature of the active ingredient no carriers might, however, be required in the case of soft gelatine capsules). Suitable carrier materials for the production of solutions and syrups are, for example, water, polyols, sucrose, invert sugar and the like. Suitable carrier materials for injection solutions are, for example, water, alcohols, polyols, glycerol and vegetable oils. Suitable carrier materials for suppositories are, for example, natural or hardened oils, waxes, fats and semi-liquid or liquid polyols. Suitable carrier materials for topical preparations are glycerides, semi-synthetic and synthetic glycerides, hydrogenated oils, liquid waxes, liquid paraffins, liquid fatty alcohols, sterols, polyethylene glycols and cellulose derivatives.
Usual stabilizers, preservatives, wetting and emulsifying agents, consistency-improving agents, flavour-improving agents, salts for varying the osmotic pressure, buffer substances, solubilizers, colorants and masking agents and antioxidants come into consideration as pharmaceutical adjuvants.
The dosage of the compounds of formula I can vary within wide limits depending on the disease to be controlled, the age and the individual condition of the patient and the mode of administration, and will, of course, be fitted to the individual requirements in each particular case. For adult patients a daily dosage of about 1 to 1000 mg, especially about 1 to 100 mg, comes into consideration. Depending on severity of the disease and the precise pharmacokinetic profile the compound could be administered with one or several daily dosage units, e.g. in 1 to 3 dosage units.
The pharmaceutical preparations conveniently contain about 1-500 mg, preferably 1-100 mg, of a compound of formula I.